(A) Human Papillomavirus Types
Human papillomaviruses (hereinafter "HPV") are recognized as a cause of various epithelial lesions such as warts, condylomas and dysplasias (see Gissmann, L., Cancer Surv., 3: 161 (1984); Pfister, H., Biochem. Pharmacol., 99: 111 (1983); Durst, M. et al, Proc. Natl. Acad. Sci. USA, 80: 3812 (1983) and Boshart, M. et al, EMBO J., 3:1151 (1984)). Dysplasias of the cervix (also known as cervical intraepithelial neoplasia (CIN)) are believed to be early events in the progression to cervical cancer; the progression proceeding from mild dysplasia (CIN I), to moderate dysplasia (CIN II), to severe dysplasia, to carcinoma in situ (collectively CIN III), to invasive cancer.
Studies examining the association of HPV type with dysplasias of the cervix and cancer of the cervix have shown that HPV types 6, 11 16, 18, 31 and 33 are associated with a high percentage of genital lesions (see Gissmann, L., Cancer Surv., 3: 161 (1984); Pfister H., Biochem. Pharmacol., 99: 111 (1983); Durst, M. et al, Proc. Natl. Acad. Sci. USA, 80: 3812 (1983); Boshart, M. et al, EMBO J., 3: 1151 (1984); de Villiers, E. -M. et al, J. Virol., 40: 932 (1981); Gissmann, L. et al, J. Virol., 44: 393 (1982); Lorincz, A. T. et al. J. Virol., 58: 225 (1986) and Beaudenon, S., Nature, 321: 246 (1986)).
HPVs are grouped into types based on the similarity of their DNA sequence. Two HPVs are taxonomically classified as being of the same type if their DNAs cross-hybridize to greater than 50%, as measured by hybridization in solution under moderately stringent hybridization conditions, which are defined as approximately 25.degree. C. below the melting temperature of a perfectly base-paired double-stranded DNA (conveniently written as T.sub.m -25.degree. C.), followed by chromatography on hydroxyapatite to separate double-stranded DNA from single-stranded DNA (see Coggin, J. R. et al, Cancer Res., 39: 545 (1979)). The melting temperature (T.sub.m) of a perfectly base-paired double-stranded DNA can be accurately predicted using the following well established formula: EQU T.sub.m =16.6.times.log [Na.sup.+ ]+0.41.times.%G:C+81.5-0.72.times.(%)(v/v) formamide.
The above formula provides a convenient means to set a reference point for determining non-stringent and stringent hybridization conditions for various DNAs in solutions having varying salt and formamide concentrations without the need for empirically measuring the T.sub.m for each individual DNA in each hybridization condition.
If less than 50% of the respective HPV DNAs are able to cross-hybridize in solution under moderately stringent conditions to form fully or partially double-stranded structures, as measured and defined by the ability to bind to hydroxyapatite, then the HPV DNAs are not sufficiently related to be taxonomically classified as being of the same type. A cut-off of 50% cross-hybridization using this method is employed as the consensus criterion for the assignment of novel HPV types for nomenclature purposes. This method for measuring the degree of cross-hybridization between HPV DNAs has been historically adopted as the method to be used to determine whether two HPV DNAs represent different isolates of a common type or represent isolates of different types. The use of this criterion pre-dates the establishment of clinical criterion for determining and defining HPV types. As discussed in more detail below, the clinical criterion for determining and defining HPV types is based upon the epidemiological distribution of HPV types among genital lesions.
The above-described method of measuring the degree of cross-hybridization is based on an assessment of the extent of formation of fully or partially double-stranded DNA molecules after the hybridization reaction. However, it should be noted that conversion of 50% of the DNAs into fully or partially double-stranded DNA molecules does not imply that the nucleotide sequences of the DNAs are 50% homologous.
As discussed above, HPVs can also be grouped into types based on clinical criterion. That is, it has been observed that HPV of different types, as defined by the degree of cross-hybridization criterion described above, show distinct epidemiological distributions among gential lesions of different severities and among different geographic populations.
For example, HPV 6 and HPV 11 are principally associated with benign lesions such as exophytic condylomas and to a lesser extent with flat condylomas (see Gissmann, L. et al, Proc. Natl. Acad. Sci., USA, 80: 560 (1983)). HPV 6 and HPV 11 are also detected in certain rare types of malignant epithelial tumors (see Zachow, K. R. et al, Nature, 300: 771 (1982) and Rando, R. F., J. Virol., 57: 353 (1986)). In contrast, HPV 16, HPV 18, HPV 31 and HPV 33 are detected with varying degrees of frequency in cervical and other anogenital cancers as well as their precursor lesions (see Durst, M. et al, Proc. Natl. Acad. Sci., USA, 80: 3812 (1983), Boshart, M. et al, Embo. J., 3: 115 (1984), Lorincz, A. T. et al, J. Virol., 58: 225 (1986) and Beaudenon, S., Nature, 321: 246 (1986)). This distribution of HPV 16, HPV 18, HPV 31 and HPV 33 is believed to reflect a greater risk of, or a more rapid progression to, cervical cancer arising from gential lesions infected with HPV 16, HPV 18, HPV 31 and HPV 33 as compared to lesions infected with HPV 6 and HPV 11. As a result, the determination of HPV types has clinical-diagnostic value, i.e., such is an important factor in the assessment of risk of cancer development in patients who exhibit evidence of HPV infection. Based on the assessed risk of cancer development, appropriate therapeutic treatments can be selected.
In addition, HPV 16 is more prevalent in Europe than in Africa (Durst, M. et al, Proc. Natl. Acad. Sci., USA, 80: 3812 (1983)), whereas HPV 18 is more prevalent in Africa than in Europe (Boshart, M. et al, EMBO J., 3: 1115 (1984)).
Accordingly, within the context of the present invention, two HPVs are considered to be of the same type if either (1) they meet the criterion for the degree of cross-hybridization discussed above or (2) if they show substantially the same epidemiological distribution of cross-hybridization among genital lesions and they both cross-hybridize with the same genital lesions which comprise the epidemiological distribution.
It has been found that a significant percentage of cervical cancer and genital lesions which have the potential to progress to cervical cancer contain "new" HPV types which do not correspond to any of the known HPV types. Thus, in light of the known association of specific HPV types with genital lesions which have a high risk of progression to cervical cancer, the ability to detect and group these "new" HPV types allow the risk of cervical cancer associated with these "new" HPV types to be ascertained in patients who exhibit evidence of HPV infection and who may be infected with these "new" HPV types.
(B) Cloning of HPV Types
In spite of long standing efforts in the art, it has not been possible to propagate HPV in cell culture in vitro. However, recombinant DNA cloning techniques have made it possible to isolate and purify the DNA of many HPV types such as HPV Types 6, 11, 16, 18, 31 and 33 (see Durst, M. et al, Proc. Natl. Acad. Sci. USA, 80: 3812 (1983); Boshart, M. et al, EMBO J., 3: 1151 (1984); de Villiers, E.-M. et al, J. Virol., 40: 932 (1981); Gissmann, L. et al, J. Virol., 44: 393 (1982); Lorincz, A. T. et al J. Virol., 58: 225 (1986) and Beaudenon, S., Nature, 321: 246 (1986)). Most of the knowledge regarding HPVs has been derived from the study of the DNA sequence in such recombinant DNAs and the use of these DNAs to prepare nucleic acid hybridization probes for detection of HPV in tissue samples.
(C) Hybridization Probes
As discussed above, HPV DNA has been employed as hybridization probes to differentiate HPV types. Two HPV DNAs of different types can be readily distinguished by hybridization under stringent hybridization conditions, which are defined as approximately 10.degree. C. below the melting temperature of a perfectly based-paired double-stranded DNA hybrid (conveniently written as T.sub.m -10.degree. C.), using such hybridization probes. Similarly, an HPV DNA of one type can be readily distinguished from an HPV RNA of another type by hybridization under stringent hybridization conditions which are defined as approximately 10.degree. C. below the melting temperature of a perfectly based-paired double-stranded DNA-RNA hybrid (conveniently written as T.sub.m -10.degree. C.), using such hybridization probes. Further, two HPV RNAs of different types can be readily distinguished by hybridization under stringent hybridization conditions, which are defined as approximately 10.degree. C. below the melting temperature of a perfectly based-paired double-stranded RNA-RNA hybrid (conveniently written as T.sub.m -10.degree. C.), using such hybridization probes. It should be noted that HPV DNAs or RNAs which are designated as different types using the above criterion, may in fact have as much as 80% of their nucleotide sequences in common.
Furthermore, two HPV DNAs of different types are able to cross-hybridize under non-stringent hybridization conditions, which are defined as approximately 35.degree. C. or more below the melting temperature of a perfectly base-paired double-stranded DNA-DNA hybrid (conveniently written as T.sub.m -35.degree. or more), using such hybridization probes. Similarly, an HPV DNA of one type is able to cross-hybridize with an HPV RNA of another type by hybridization under non-stringent hybridization conditions which are defined as approximately 35.degree. C. or more below the melting temperature of a perfectly based-paired double-stranded DNA-RNA hybrid (conveniently written as T.sub.m -35.degree. C. or more), using such hybridization probes. Further, two HPV RNAs of different types are able to cross-hybridize under non-stringent hybridization conditions, which are defined as approximately 35.degree. C. or more below the melting temperature of a perfectly based-paired double-stranded RNA-RNA hybrid (conveniently written as T.sub.m -35.degree. C. or more), using such hybridization probes (see Anderson, L. M. et al, Nucleic Acid Hybridization, pages 73-111, Eds. B. D. Hames and S. J. Higgins, I.R.L. Press, Oxford, England and Washington, D.C., USA (1985)).
The melting temperatures of DNA-DNA, DNA-RNA and RNA-RNA hybrids of the same nucleotide sequences may be different in various chemical environments. The effect of various compounds on the relative melting temperatures of these various hybrids has been studied for several agents. For example, it is well known that increasing the concentration of formamide differentially destabilizes DNA-DNA hybrids more than DNA-RNA hybrids so that at high concentrations of formamide, such as 80% (v/v), a DNA-RNA hybrid may have a significantly higher melting temperature than a DNA-DNA hybrid of the same nucleotide sequence.
As discussed above, the melting temperature of a DNA-DNA hybrid can be predicted as described in Anderson, L. M. et al, Nucleic Acid Hybridization, pages 73-111, Eds. B. D. Hames and S. J. Higgins, I.R.L. Press, Oxford, England and Washington, D.C., USA (1985)). Further, the melting temperature of a DNA-DNA hybrid can be empirically determined as described in Howley, P. et al, J. Biochem., 254: 4876 (1979). The melting temperature of a DNA-RNA hybrid and a RNA-RNA hybrid can also be determined by means well known in the art.
Thus, it is possible to test a tissue sample for the presence of HPV DNA or RNA in general and/or a particular HPV DNA or RNA type by nucleic acid hybridization depending upon what conditions, i.e., stringent or non-stringent, are employed for hybridization.